it is a chapter of microbiology

1. CULTURE MEDIA
B Y P I Y U S H
B . S C . ( H O N S . ) N U R S I N G

2. INTRODUCTION
• To identify clinically important bacteria, they need to be isolated from the samples of submitted
to the laboratory.
• This is done by inoculating the samples on growth media required for the bacteria to replicate and
form colonies on solid media or suspensions in liquid media.
• This forms the starting point in the identification and antibiotic susceptibility testing of the
organism.

3. TYPES OF CULTURE MEDIA
LIQUID MEDIA: Liquids are used to obtain bacterial growth from blood or water when large
volumes have to be tested and for preparing bulk cultures of antigens or vaccines.
 Bacteria grow diffusely in liquids.They produce discrete, visible growth on solid media.
 If inoculated in suitable dilutions, bacteria form colonies, which are clones of cells originating from a
single bacterial cell. Fluid enrichment media are first incubated at 370C and then sub- cultured on a
solid medium to get individual isolated colonies.
 SOLID MEDIA: On solid media, bacteria have distinct colony morphology and exhibit many
characteristic features such as pigmentation or hemolysis, making identification easy.
 Agar or agar- agar is used to prepare solid media.
 Agar is obtained from a type of seaweed. It has virtually no nutritive value and is not affected by the
growth of bacteria.
 It melts at 98℃ and usually sets at 42℃ depending on the agar concentration.
 Approximately 2% of agar is used for solid media. Another ingredient of common media is
peptone.

4. • Peptone is a complex mixture of partially digested proteins. Its constituents are proteoses,
polypeptides and amino acids, a variety of inorganic salts including phosphates, potassium and
magnesium and certain accessory growth factors such as riboflavin.
• Blood, serum and yeast extract are other common ingredients of media.
 SIMPLE MEDIA: Also known as basal media. It consist of meat extract, sodium chloride and
water. Nutrient agar, made by adding 2% agar to nutrient broth, is the simplest and most common
medium in routine diagnostic laboratories.
 COMPLEX MEDIA: These have added ingredients for special purposes or for bringing out
certain characteristics or for providing special nutrients required for the growth of the bacterium
under study.
SYNTHETIC MEDIA: These media are prepared from pure chemical substances and the
exact composition of the medium is fully documented.These are used for various special studies
such as metabolic requirements.
ENRICHED MEDIA:To cultivate bacteria with exacting nutritional requirements,
substances such as blood serum or egg are added to basal medium. Examples are:
blood agar, chocolate agar and media containing egg.

5. BRAIN- HEART INFUSION BROTH
(BHIB)
• This is a highly nutritious, buffered fluid culture medium prepared by non-
enzymic infusion from calf brain and cow heart, often with peptone and
dextrose added.
• It is suitable for the cultivation of fastidious organisms.

6. BLOOD AGAR
• This is a solid- culture medium consisting of agar, peptones and blood.
• The blood is usually from sheep, but horse, cow and pig blood may be used. Blood agar
support the growth of most aerobic and anaerobic bacteria.
• Vitamin k, cysteine and hemin supplementation enhances the growth of anaerobic bacteria.
• Blood agar can indicate the degree of hemolysis caused by hemolysin.
• Based on this. It is used to differentiate among Gram- positive cocci. Hence, it is also known as
a differential medium.

7. • Beta Hemolysis: It refers to complete lysis of the red blood cells and hemoglobin; thus
results incomplete clearing of the blood agar medium surrounding the colonies. E.g. Group A
Steptoccoci.
• Alpha Hemolysis: It refers to the partial lysis of red blood cells and hemoglobin; this results
in greenish discolouration of the blood agar around the colonies . E.g.:Viridans Streptococci
• No hemolysis results in no change of the blood agar medium. E.g; Enterococci.

8. CHOCOLATE AGAR
• This is made by heating a mixture of sheep blood and nutrient agar, hemoglobin and the related
substance hemin( also called X factor) and nicotinamide adenine dinucleotide ( NAD, also calledV
factor) these are released during the process of heating .
• The medium is called ‘chocolate’ agar because of its colour.
• Chocolate agar is used to grow fastidious organisms, including H. influenza,N. meningitidis and N.
gonorrheae and Pneumococcus.

9. ENRICHMENT MEDIA
• These media are used to suppress commensal bacteria while allowing the pathogen to remain
viable and grow.
• It is employed for specimen with mixed flora. E.g: fecal sample to isolate diarrheagenic bacteria.
• Substances that have stimulating effect on the bacteria to be grown or an inhibitory effect on
those to be suppressed are incorporated in the medium.
• Examples:Tetrathionate broth where the tetrathionate inhibits coliforms while allowing
typhoid- parathyroid bacilli to grow and Selenite F broth for the bacteria which cause
dysentery.

10. SELECTIVE MEDIA
• The inhibiting substance is added to a solid medium, it enables a greater number of the
required bacterium to form colonies than the other bacteria.
• For example: Desoxycholate Citrate Agar for fecal samples andThiosulphate Citrate Bile
Sucrose agar forVibrio species in cholera.
• The alkaline Ph isolatesVibrio. Organisms that ferments sucrose appear yellow, while those
that do not, appear green, thus acting as an indicator medium too.

11. THAYER- MARTIN MEDIUM
• THEThayer- Martin medium containing antimicrobials ( vancomycin 3.0mg, colistin 7.5mg and
nystatin 12.5 units per ml of agar) in chocolate agar is used to isolate N. gonorrhoeae from
clinical specimens.
• The antimicrobials suppress the growth of other commensal organisms which may inhibit the
growth of N. gonnorrhoeae.Thayer- Martin plates are incubated in an atmosphere containing
3-10% C𝑂2.

12. LOWESTEIN- JENSEN MEDIUM
• This medium is used for primary isolation of Mycobacterium species.
• It consists of mineral salts, asparagine, glycerol, malachite green and hen’s eggs.
• The medium is sterilized by inspissation .
• The malachite green prevents the growth of the most other microorganisms.
• This medium is used since it will support the growth of a very small inoculum.

13. INDICATOR MEDIA
• These media contain an indicator that changes colour when a bacterium grows in them, e.g.,
sulphite inWilson- Blair medium.
• S. typhi reduces sulphite to sulphide to give a black metallic sheen on the colony.
• Potassium tellurite in Mcleod’s medium is reduced to metallic tellurium by corynebacterium
diphtheriae to produce black colonies.

14. DIFFERENTIAL MEDIA
• The MacConkey medium which consists of peptone, lactose, agar, neutral red and taurocholate
shows lactose fermenters as pink colonies, while non- lactose fermenters are colourless or
pale.This may be termed indicator medium.
• Many facultative anaerobes in the intestine are lactose fermenters and are pink in colour.
• Several well- known pathogens do not ferment lactose and are colourless (Shigella- Salmonella
species)
• There are many special media for demonstrating particular characteristics like, Nagler’s
medium which enables us to view lecithinase activity.

15. TRANSPORT MEDIA
• Special media are devised for transporting specimens suspected to have fastidious organisms.
• These are termed transport media, e.g., Stuart’s medium – anon nutrient, soft agar gel
containing a reducing agent to prevent oxidation and charcoal to neutralize certain bacterial
inhibitors- is used to transport specimen for isolation of gonococci, and buffered glycerol saline
for enteric bacilli.

16. ANAEROBIC BACTERIA
o Anaerobic media includes Robertson’s cooked meat medium and thioglycollate medium.
o These media are used to grow anaerobic organisms in presence of reducing substances or
absence of oxygen.
THIOGLYCOLLATE MEDIUM
 This medium supports the growth of all organisms with varied oxygen requirements:
anaerobes, aerobes and facultative anaerobes.
 An oxygen indicator turns the medium pink or blue at the top of the tube.
 This medium is boiled before use to eliminate oxygen which is less soluble at hot
temperatures.
 Obligate aerobes grow only at the top of the tube of medium, microaerophiles in the middle,
while anaerobes grow only at bottom.
 The medium contains yeast extract ; casitone, sodium chloride, l-cysteine; thioglycollic acid;
agar, methylene blue and deionized water at a final ph of 7.2

17. MEDIA FOR FUNGUS CULTURE
 SABOURAUD DEXTROSE AGAR:This culture medium permits the growth of yeasts and
most filamentous fungi.
• It has a high concentration of either glucose or maltose and also contains mycological peptone.
• The medium has a low ph ( about 5.0), which inhibits the growth of most bacteria.
• Antibacterial agents ( chloramphenicol 40mg or gentamicin 50mg)per litre of medium) can also be
added to augment the antibacterial effect.
INCUBATION OF CULTURE MEDIA: Culture plates are incubated fo a minimum of 48
hours at 37°C for bacteria and 22°C and 30°C for fungi.
• If a microaerophilic bacterium is suspected, then that growth condition should also be
included.
• Both bacteria and fungi grow better in 55 carbon dioxide than in air alone.
• Anaerobic plates should be incubated in an anaerobic cabinet for 7 or 14 days.

18. PotassiumTellurite Agar
Sabouraud Dextrose Agar

19. MEDIA FOR SPECIAL USE
• Media for antibiotic susceptibility testing is cation adjusting Mueller – Hinton agar( CAMHA)
• Media incorporated with enzymes( e.g., Betalactamase detection) to detect antibiotic
resistance.
• Screen agars to select out MRSA and vancomycin resistance in staphylococci.
• CHROM agar to speciate candida depending upon the colour produced by the species.

20. CLASSIFICATION OF CULTURE MEDIA
TYPE OF CULTURE MEDIA EXAMPLES
LIQUID Brain- heart infusion broth, peptone water, nutrient broth
SOLID Nutrient agar, blood agar, chocolate agar
SIMPLE Non- nutrient agar, nutrient agar
COMPLEX Thiosulphate Citrate bile salt sucrose agar.
SYNTHETIC OR DEFINED Hank’s balanced salt solution

21. BASED ON FUNCTIONAL
REQUIREMENT
TYPE OF CULTURE MEDIA EXAMPLES
ENRICHED Todd-Hewitt broth
ENRICHMENT Selenite F medium
SELECTIVE Salmonella Shigella agar
INDICATOR MacConky agar
DIFFERENTIAL Mannitol Salt agar
TRANSPORT Stuart’s transport agar